CN114235781A - Method for quantitatively detecting beta-galactosidase in seawater based on surface enhanced Raman spectroscopy technology - Google Patents

Method for quantitatively detecting beta-galactosidase in seawater based on surface enhanced Raman spectroscopy technology Download PDF

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CN114235781A
CN114235781A CN202111583690.4A CN202111583690A CN114235781A CN 114235781 A CN114235781 A CN 114235781A CN 202111583690 A CN202111583690 A CN 202111583690A CN 114235781 A CN114235781 A CN 114235781A
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方家松
吴勇
张宏鸽
包天强
曹军伟
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    • G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase

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Abstract

The invention discloses a method for quantitatively detecting beta-GAL based on a surface enhanced Raman spectroscopy technology, which comprises the following steps: a. respectively taking 180 mu L of BCIG solution, beta-GAL solution and DMSO solution; b. obtaining a plurality of beta-GAL samples with different activities in advance; c. dropwise adding equal volume of gold nano sol particles into a plurality of different active standard solutions, and respectively carrying out SERS detection; d. after a seawater sample to be detected and BCIG are subjected to incubation reaction, adding DMSO and surface-enhanced gold nano sol particles, and directly detecting SERS signals of a product and the DMSO; e. and d, comparing the SERS signal obtained in the step d with a standard curve to obtain the activity of the sample to be detected. The method has good reliability, and the DMSO is introduced as an internal standard substance, so that the stability of detection is improved; the method has the advantage of no need of sample pretreatment; provides powerful scientific theoretical support for in-situ quantitative detection of the activity of the extracellular enzymes in seawater.

Description

Method for quantitatively detecting beta-galactosidase in seawater based on surface enhanced Raman spectroscopy technology
Technical Field
The invention belongs to the technical field of Raman spectrum detection, and particularly relates to a method for quantitatively detecting beta-galactosidase in seawater based on a surface enhanced Raman spectrum technology.
Background
The ocean covers 71% of the earth's surface area, and the marine ecosystem is the largest ecosystem on earth. In the marine environment, mostly Dissolved Organic Matter (DOM) generated by primary productivity and DOM of other sources exist in the form of high molecular organic matter, for which microorganisms cannot directly utilize, and only after extracellular enzymes decompose the high molecular polymers into small molecular monomers or polymers with molecular weight less than 700 daltons (Dalton, Da) can be absorbed by the microorganisms. In this process, marine microorganisms are the major players, and the enzymatic hydrolysis process of organics mediated by them is key to limit the interconversion of DOM and Particulate Organics (POM). In the hydrolysis process, the released monomeric compounds can also be utilized by the microorganisms themselves to maintain metabolism, which serves both as decomposers and producers, and as beneficiaries. Therefore, the activity of microbial extracellular enzymes is the core driver of the whole process, and plays a decisive role in the aspects of the circulation rate, the spatial distribution and the like of ocean carbon. The research of the marine exoenzyme has important significance for comprehensively understanding the material circulation, the energy circulation and the life activities of microorganisms of a marine ecosystem and evaluating the water quality and the ecological efficiency.
Beta-galactosidase (known as beta-D-galactosidase, beta-GAL) is a hydrolase that specifically hydrolyzes beta-1, 4-glycosidic bonds, hydrolyzing lactose to glucose and galactose. It is widely present in marine water, wherein marine plants, bacteria, fungi, etc. can produce beta-GAL, and release it to surrounding water, and decompose the polysaccharide in the marine water into monosaccharide or small molecular polysaccharide less than 700Da for maintaining self metabolism. Metabolism and death of these organisms can also provide a large amount of Organic Carbon (OC) sources for surrounding water bodies, so that the activity of the beta-GAL not only reflects the marine microorganism population structure and the distribution of marine organic nutrients, but also has important significance for uncovering the mechanism of marine carbon cycle driven by marine microorganisms. Common detection methods for the activity of the beta-GAL in seawater comprise a fluorescence simulated substrate method, a spectrophotometer method and the like, and the methods have complex sample pretreatment and consume much time for detection. However, the activity of the extracellular enzyme is very susceptible to various factors including temperature, pressure, pH, oxygen content, etc., so a rapid and efficient method for detecting the activity of the extracellular enzyme is needed.
The laser Raman spectrum is an inelastic scattering phenomenon caused by energy exchange between laser photons and molecules of a substance due to the fact that the laser Raman spectrum irradiates the surface of the substance, and can reflect the internal energy level structure of the molecules of the substance and represent molecular vibration information. Surface Enhanced Raman Spectroscopy (SERS) utilizes the optical enhancement effect of metal nanoparticles such as gold and silver to enhance the Raman spectrum signal of target molecules adsorbed on the particles, thereby realizing rapid detection of low-concentration substances. In recent years, the method has the advantages of rapidness, simple sample pretreatment, no damage, no contact and the like, and is widely applied to the fields of food safety, biological detection and the like.
Disclosure of Invention
In order to overcome the problems, the invention provides a method for quantitatively detecting beta-galactosidase in seawater based on a surface enhanced Raman spectroscopy technology.
The method for quantitatively detecting the beta-galactosidase in the seawater based on the surface enhanced Raman spectroscopy technology comprises the following steps:
a. respectively taking 180 mu L of BCIG solution, beta-GAL solution, DMSO solution, solution obtained after the reaction of BCIG and beta-GAL, adding 180 mu L of DMSO solution for dissolving, adding 200 mu L of gold nanoparticles (70nm), and performing on-machine detection on respective surface enhanced Raman spectra; (the surface enhanced Raman spectra of the solvent and the substrate used in the experiment are used as a blank group, and the experiment proves that 600cm-1The Raman spectrum peak at the wave number is the characteristic peak of the product, 677cm-1The Raman spectrum peak at the wave number is a DMSO characteristic peak and can be used as an internal standard peak of the experiment)
b. Pre-obtaining a plurality of beta-GAL samples with different activities, respectively mixing and incubating the beta-GAL samples with BCIG solution for a period of time, and adding DMSO solution as standard solution; (preparing beta-GAL solutions with different activities and establishing a quantitative standard curve. the action of the DMSO solution can dissolve products on one hand to ensure that the solution is more uniform so as to improve the stability and reliability of detection, and can be used as an internal standard substance to ensure that the quantitative model is more stable on the other hand)
c. Respectively dripping equal-volume surface enhanced gold nano sol particles into a plurality of different beta-GAL active standard solutions, respectively carrying out SERS detection, and then drawing a standard curve according to the relation between the obtained SERS signals of the different active standard solutions and the relative strength of the SERS signals of DMSO and the activity logarithm value of the standard solutions; (enhancing the intensity of the target peak of the product and the internal peaks of DMSO, establishing a quantitative standard curve according to the relationship between the target peak and the internal peaks)
d. After a seawater sample to be detected (usually the seawater contains beta-GAL) and BCIG are incubated and reacted, DMSO and surface enhanced gold nano sol particles are added, and SERS signals of a product and the DMSO are directly detected; (verification that the sensitivity requirement of the method can reach the requirement of detecting the beta-GAL activity in seawater)
e. And d, comparing the SERS signal obtained in the step d with a standard curve to obtain the activity of the sample to be detected. (use the above-mentioned standard curve to detect the sea water sample beta-GAL activity quantitatively)
Further, the particle size of the gold nanoparticles added in the step a is 70nm in 200 mu L.
Further, the SERS signal of DMSO in the step c and the step d is selected to be that DMSO is 677cm-1Peak high intensity at raman shift.
Further, the SERS signal of the beta-GAL in the step c and the step d is that the beta-GAL is selected to be 600cm-1The peak intensity at the Raman shift, steps d and e are aimed at detecting the activity of the beta-GAL in the seawater, on one hand, we need to test to verify whether the beta-GAL exists in the seawater sample, and on the other hand, the obtained standard curve is used for the quantitative activity analysis of the beta-GAL in the seawater sample, so that the beta-GAL does not appear.
Furthermore, the average relative standard deviation of the enzyme activity data obtained by the beta-GAL enzyme activity standard solution is less than 15%, and the detection method has good stability.
Further, the average value of the enzyme activity data obtained by the beta-GAL enzyme activity standard solution is 600cm-1/677cm-1。
Further, fitting of the standard curve of step cThe standard equation is that y is 0.784 x +0.004, and the correlation coefficient R2＝0.936。
Has the advantages that:
(1) the activity of the beta-GAL in the seawater is detected by using a laser Raman spectrum technology for the first time, and the sensitivity reaches the requirement of detecting the beta-GAL in the seawater;
(2) the average relative standard deviation of the method is less than 15%, the method has good reliability, and DMSO is introduced as an internal standard substance, so that the stability of detection is improved;
(3) the method has the advantages of no need of sample pretreatment (only adding a substrate for reaction for a period of time, then adding an internal standard and surface-enhanced gold nanoparticles for direct detection), no damage (exciting light is visible light, no damage is caused to the sample), and rapid detection (compared with a fluorescence method which requires detection time of dozens of hours or even dozens of hours, the method only needs several hours and is more suitable for in-situ detection);
(4) the method provides powerful scientific theoretical support for in-situ quantitative detection of the activity of the extracellular enzymes in the seawater.
Drawings
FIG. 1 is a reaction scheme for the hydrolysis of BCIG by β -GAL;
FIG. 2 is a surface enhanced Raman spectrum of an experimental solvent and substrate;
FIG. 3 is a surface enhanced Raman spectrum corresponding to different β -GAL activities;
FIG. 4 is a linear equation fit of a standard curve;
FIG. 5 is a graph of SERS after reaction of a seawater sample with BCIG.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
Detection principle of the invention
The beta-GAL can carry out specific catalytic hydrolysis on a beta-1, 4-glycosidic bond, the hydrolysis principle of the beta-GAL is shown in figure 1, the beta-GAL is hydrolyzed by 5-bromo-4-chloro-3-indole-beta-D-galactoside (BCIG) without SERS characteristic to obtain 5-bromo-4-chloro-3-indole (BCI), a water-insoluble BCI oxidized dimer is formed through rapid oxidation, the oxidized dimer has stronger SERS characteristic, and the ALP activity is determined by establishing the relationship between different ALP activities and the SERS characteristic peak intensity of a product.
Theoretically, the position of the Raman spectrum characteristic peak of the BCI oxidized dimer of the product is 600cm-1In the vicinity of the wave number, it was verified by the following experiment.
Respectively taking 200 mu L and 2mg/mL BCIG solution, 200 mu L and 5U/L beta-GAL solution, 200 mu L DMSO solution, and BCIG (20 mu L and 2mg/mL) and beta-GAL (180 mu L and 5U/L) solution after reacting for 4h, respectively adding 200 mu L gold nanoparticle colloid into 4 upper sample bottles, fully mixing uniformly, and detecting SERS (excitation wavelength of 785nm, excitation time of 10s, excitation frequency of 5mW, each sample is collected for 5 times of Raman spectrum).
As shown in FIG. 2, BCIG solution and beta-GAL solution have no SERS characteristics, and DMSO solution is 600cm-1Near wavenumber, no Raman spectrum peak, which is 677cm-1And 700cm-1Two obvious SERS characteristic peaks near the wave number respectively represent a C-S-C symmetric stretching vibration peak and a C-S stretching vibration peak, and 677cm is selected-1The Raman spectrum peak at wavenumber is used as an internal standard peak for quantitative analysis of extracellular enzyme activity. Wherein SERS generated after BCI oxidized dimer is generated by 4 hours of reaction of BCIG and beta-GAL is 600cm-1And a strong Raman peak appears at the wave number, and the Raman peak is caused by plane vibration of C ═ C-CO-C in the chemical structure of the product, namely the characteristic peak of the product.
Example 1 quantitative model
5 900 μ L beta-GAL solutions with different activities (50U/L, 10U/L, 5U/L, 1U/L, 0.5U/L) are mixed and incubated with 2mg/mL BCIG solution and 100 μ L BCIG solution for 4h, 180 μ L reaction solution is added into 20 μ L DMSO solution, and then the SERS signals are respectively measured, and the obtained spectrum is shown in FIG. 3.
As can be seen in FIG. 3, there is no direct linear relationship between SERS characteristic peak intensity and ALP concentration of the product BCI oxidized dimer, since the intensity of the Raman spectrum is stabilized by laser power, enhancing reagent uniformityAnd background noise of a solvent, and the like, and it is difficult to directly perform quantitative analysis by using the intensity of a characteristic peak of a Raman spectrum. Thus, the DMSO solvent was added at 677cm-1And (3) taking the Raman spectrum characteristic peak near the wave number as an internal standard peak, and establishing a quantitative detection model by using an internal standard method to realize the quantitative detection of the beta-GAL. Table 1 shows SERS characteristic peak intensity information for the substrate and internal standard.
Table 1: characteristic peak intensities of substrate and internal standard
Figure BDA0003427691230000071
As can be seen from Table 1, overall, the characteristic peak of the product (600 cm)-1) Intensity and internal standard peak (677 cm)-1) The intensity gradually decreases with decreasing β -GAL activity, but there is no good functional relationship between them. RSD of the intensity ratio corresponding to each enzyme activity is less than 10%, which indicates that the reliability of SERS data is high. beta-GAL concentration and SERS intensity ratio (600 cm) using least squares-1/677cm-1) A linear fit is performed. As shown in fig. 4.
DMSO solvent was introduced as an internal standard at 677cm-1The characteristic peak at the wave number is taken as an internal standard peak, the logarithm of 5 beta-GAL activities of 50U/L, 10U/L, 5U/L, 1U/L and 0.5U/L is taken as the abscissa, and the ordinate is the characteristic peak of the product (600 cm)-1) And internal standard peak (677 cm)-1) The ratio, the fitting standard equation is: y 0.784 x +0.004, correlation coefficient R20.936,. beta. -GAL activity/Raman spectrum characteristic peak intensity ratio (600 cm)-1/677cm-1) Shows a strong linear relationship. This model has the ability to quantitatively detect β -GAL activity.
Example 2 sea water verification test
Samples of fresh seawater were taken from the east China sea (30 ° 39 '48 "N, 122 ° 29' 48" E) in 12 months of 2020. The sample is ocean surface seawater, and the fishing boat directly samples. mu.L of fresh seawater sample and 2mg/mL and 100 mu.L of BCIP solution are mixed and incubated for 4h, 180 mu.L of reaction solution is added into 20 mu.L of DMSO solution, and then SERS signals are measured, and the obtained spectrum is shown in FIG. 5.
As shown in FIG. 5, 600cm is shown-1The obvious Raman spectrum peak (the peak intensity is 16779) appears, which indicates that the method is used to successfully and qualitatively detect the existence of beta-GAL (beta-GAL) in seawater, 677cm-1The Raman spectrum intensity at the wave number reaches 17550, the ratio of two peak values is 0.956 due to the Raman spectrum peak caused by C-S-C symmetric stretching vibration in DMSO, the value is substituted into the model to realize the quantitative detection of the beta-GAL activity of the seawater sample, and the obtained commercial beta-GAL activity of the water sample, which is equivalent to 0.824U/L, is obtained.
BCIG is taken as a substrate, DMSO is taken as an internal standard substance, and a quantitative detection method for detecting beta-GAL activity in seawater based on SERS is provided. The results show that the beta-GAL activity and the ratio of the characteristic peak to the internal standard peak intensity (600 cm)-1/677cm-1) The model has good linear relation, the correlation coefficient is 0.936, and the beta-GAL activity in the seawater sample is successfully and quantitatively detected by using the model, so that the rapid detection of the beta-GAL activity in the seawater is realized. Meanwhile, the method can also be applied to the detection of the activity of other microbial exoenzymes in the seawater, and lays a solid scientific foundation for the in-situ detection of the activity of the microbial exoenzymes in the seawater.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Claims (7)

1. The method for quantitatively detecting the beta-galactosidase in the seawater based on the surface enhanced Raman spectroscopy technology is characterized by comprising the following steps:
a. respectively taking 180 mu L of BCIG solution, beta-GAL solution, DMSO solution, solution obtained after the reaction of BCIG and beta-GAL, adding 180 mu L of DMSO solution for dissolving, adding 200 mu L of gold nanoparticles, and detecting respective surface enhanced Raman spectra on a machine;
b. pre-obtaining a plurality of beta-GAL samples with different activities, respectively mixing and incubating the beta-GAL samples with BCIG solution for a period of time, and adding DMSO solution as standard solution;
c. respectively dripping equal-volume surface enhanced gold nano sol particles into a plurality of different beta-GAL active standard solutions, respectively carrying out SERS detection, and then drawing a standard curve according to the relation between the obtained SERS signals of the different active standard solutions and the relative strength of the SERS signals of DMSO and the activity logarithm value of the standard solutions;
d. after a seawater sample to be detected and BCIG are subjected to incubation reaction, adding DMSO and surface-enhanced gold nano sol particles, wherein the seawater usually contains beta-GAL, and directly detecting SERS signals of a product and the DMSO;
e. and d, comparing the SERS signal obtained in the step d with a standard curve to obtain the activity of the sample to be detected.
2. The method for quantitatively detecting the beta-galactosidase in the seawater based on the surface-enhanced Raman spectroscopy technology of claim 1, wherein the particle size of the 200 μ L gold nanoparticles added in the step a is 70 nm.
3. The method for quantitatively detecting beta-galactosidase in seawater based on the surface-enhanced Raman spectroscopy technology of claim 1, wherein the SERS signals of DMSO in the steps c and d are selected from DMSO at 677cm-1Peak high intensity at raman shift.
4. The method for quantitatively detecting beta-galactosidase in seawater based on the surface-enhanced Raman spectroscopy (SERS) of claim 1, wherein the SERS signal of beta-GAL in the steps c and d is obtained by selecting the beta-GAL at 600cm-1Peak high intensity at raman shift.
5. The method for quantitatively detecting the beta-galactosidase in the seawater based on the surface-enhanced Raman spectroscopy technology of claim 1, wherein the average relative standard deviation of the beta-GAL enzyme activity standard solution obtained enzyme activity data is less than 15%, and the detection method has good stability.
6. The quantitative detection method based on the surface enhanced Raman spectroscopy technology according to claim 1The method for preparing beta-galactosidase in seawater is characterized in that the average value of the enzyme activity data obtained by the beta-GAL enzyme activity standard solution is 600cm-1/677cm-1。
7. The method for quantitatively detecting beta-galactosidase in seawater based on the surface-enhanced raman spectroscopy technique of claim 1, wherein the fitting standard equation of the step c standard curve is y-0.784 x +0.004, and the correlation coefficient R is2＝0.936。
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